-Tubulin is the main protein involved in the nucleation of microtubules in all eukaryotes. It forms two different complexes with proteins of the GCP family (-tubulin complex proteins): -tubulin small complexes (TuSCs) that contain -tubulin, and GCPs 2 and 3; and -tubulin ring complexes (TuRCs) that contain multiple TuSCs in addition to GCPs 4, 5 and 6. Whereas the structure and assembly properties of TuSCs have been intensively studied, little is known about the assembly of TuRCs and the specific roles of GCPs 4, 5 and 6. Here, we demonstrate that two copies of GCP4 and one copy each of GCP5 and GCP6 form a salt (KCl)-resistant sub-complex within the TuRC that assembles independently of the presence of TuSCs. Incubation of this sub-complex with cytoplasmic extracts containing TuSCs leads to the reconstitution of TuRCs that are competent to nucleate microtubules. In addition, we investigate sequence extensions and insertions that are specifically found at the N-terminus of GCP6, and between the GCP6 grip1 and grip2 motifs. We also demonstrate that these are involved in the assembly or stabilization of the TuRC.

-管蛋白是所有真核生物中微管成核的主要蛋白质。它与GCP家族蛋白（微管蛋白复合蛋白）形成两种不同的复合物：-包含-微管蛋白的微管蛋白小复合物（TuSC），以及GCP 2和3；-和微管环复合物（TuRC）除GCP 4、5和6外还包含多个TuSC。尽管已经对TuSC的结构和组装性质进行了深入研究，但对TuRC的组装和GCP的具体作用知之甚少4 ，5和6。在这里，我们证明了GCP4的两个副本以及GCP5和GCP6的一个副本分别在TuRC中形成了耐盐（KCl）的亚复合物，该复合物独立于TuSC的存在而组装。将该亚复合物与含有TuSC的细胞质提取物一起孵育，会导致能够使微管成核的TuRC重构。此外，我们研究了在GCP6的N端以及GCP6的抓地力1和抓地力2基序之间特别发现的序列延伸和插入。我们还证明了这些与TuRC的组装或稳定有关。